The clinical relevance of an inflamed appendix in Crohn's disease.

BACKGROUND AND AIMS: An appendectomy for appendiceal inflammation has been suggested to ameliorate the clinical course of patients with ulcerative colitis (UC). In contrast, for Crohn's disease (CD) an inverse association has been suggested with a higher incidence of CD and worse prognosis after appendectomy. The aim of this study was to analyse the clinical relevance of an inflamed appendix in CD patients undergoing ileocoecal resection (ICR). METHODS: All consecutive patients undergoing primary ICR between 2007 and 2018 were considered for inclusion. Microscopic data of available appendiceal resection specimens (n=99) were revised by a dedicated IBD-pathologist and scored as inflamed or not inflamed. Eighteen patients had a previous appendectomy. Pathological findings were correlated with disease characteristics and recurrence rates (clinical, endoscopic and intervention-related). RESULTS: In total, 117 patients were included: 77 (65.8%) females with a median age of 30 years [IQR 24 - 43] with a median follow up of 102 months [IQR 76-114]. Of patients without previous appendectomy (n=99), 39% had an inflamed appendix. No significant differences in disease characteristics (e.g. disease location, behaviour, time to surgery) or prognosis could be demonstrated between the two groups. In contrast, previous appendectomy (n=18) was associated with penetrating disease and numerically shorter disease duration at the time of resection. Furthermore, a trend was seen towards a stronger association with postoperative recurrence. CONCLUSION: The current study could not confirm a different prognosis for CD patients with and without an inflamed appendix. In contrast, in patients with a previous appendectomy a trend was seen towards increased postoperative recurrence, which might be related to the higher incidence of penetrating disease.

Peripheral blood DNA methylation signatures and response to tofacitinib in moderate-to-severe ulcerative colitis.

INTRODUCTION: Predictive biomarkers for treatment efficacy of ulcerative colitis (UC) treatments are lacking. Here, we performed a longitudinal study investigating the association and potential predictive power of genome-wide peripheral blood (PB) DNA methylation signatures and response to tofacitinib treatment in UC. METHODS: We recruited moderate-to-severe UC patients starting tofacitinib treatment and measured PB DNA methylation profiles at baseline (T1), after 8 weeks (T2), and in a subset (n=8), after a median of 20 weeks (T3) using the Illumina Infinium HumanMethylation EPIC BeadChip. After 8 weeks, we categorized responders (R) from non-responders (NR) based on a centrally read endoscopic response (decrease in endoscopic mayo score ≥1 or UCEIS ≥2) combined with corticosteroid-free clinical- and/or biochemical response. T1 PB samples were used for biomarker identification, while T2 and publicly available intra-class correlation (ICC) data were used for stability analyses. RNA-sequencing was performed to understand the downstream effects of the predictor CpG loci. RESULTS: In total, 16 R and 15 NR patients with a median disease duration of 7 (4-12) years and overall comparable patient characteristics at baseline were analyzed. We identified a panel of 53 differentially methylated positions (DMPs) associated with response to tofacitinib (AUROC 0.74). Most DMPs (77%) demonstrated both short- and long-term hyper stability (ICC ≥0.90), irrespective of inflammatory status. Gene expression analysis showed lower FGFR2 (pBH=0.011) and LRPAP1 (pBH=0.020), and higher OR2L13 (pBH=0.016) expression at T1 in R compared to NR. CONCLUSION: Our observations demonstrate the utility of genome-wide PB DNA methylation signatures to predict response to tofacitinib.

Systematic Review and Meta-analysis of Peripheral Blood DNA Methylation Studies in Inflammatory Bowel Disease.

BACKGROUND AND AIMS: Over the past decade, the DNA methylome has been increasingly studied in peripheral blood of inflammatory bowel disease [IBD] patients. However, a comprehensive summary and meta-analysis of peripheral blood leukocyte [PBL] DNA methylation studies has thus far not been conducted. Here, we systematically reviewed all available literature up to February 2022 and summarized the observations by means of meta-analysis. METHODS: We conducted a systematic search and critical appraisal of IBD-associated DNA methylation studies in PBL using the biomarker-based cross-sectional studies [BIOCROSS] tool. Subsequently, we performed meta-analyses on the summary statistics obtained from epigenome-wide association studies [EWAS] that included patients with Crohn's disease [CD], ulcerative colitis [UC] and/or healthy controls [HC]. RESULTS: Altogether, we included 15 studies for systematic review. Critical appraisal revealed large methodological and outcome heterogeneity between studies. Summary statistics were obtained from four studies based on a cumulative 552 samples [177 CD, 132 UC and 243 HC]. Consistent differential methylation was identified for 256 differentially methylated probes [DMPs; Bonferroni-adjusted p ≤ 0.05] when comparing CD with HC and 103 when comparing UC with HC. Comparing IBD [CD + UC] with HC resulted in 224 DMPs. Importantly, several of the previously identified DMPs, such as VMP1/TMEM49/MIR21 and RPS6KA2, were consistently differentially methylated across all studies. CONCLUSION: Methodological homogenization of IBD epigenetic studies is needed to allow for easier aggregation and independent validation. Nonetheless, we were able to confirm previous observations. Our results can serve as the basis for future IBD epigenetic biomarker research in PBL.

Long-term Temporal Stability of Peripheral Blood DNA Methylation Profiles in Patients With Inflammatory Bowel Disease.

BACKGROUND & AIMS: There is great current interest in the potential application of DNA methylation alterations in peripheral blood leukocytes (PBLs) as biomarkers of susceptibility, progression, and treatment response in inflammatory bowel disease (IBD). However, the intra-individual stability of PBL methylation in IBD has not been characterized. Here, we studied the long-term stability of all probes located on the Illumina HumanMethylation EPIC BeadChip array. METHODS: We followed a cohort of 46 adult patients with IBD (36 Crohn's disease [CD], 10 ulcerative colitis [UC]; median age, 44 years; interquartile range [IQR] 27-56 years; 50% female) that received standard care follow-up at the Amsterdam University Medical Centers. Paired PBL samples were collected at 2 time points with a median of 7 years (range, 2-9 years) in between. Differential methylation and intra-class correlation (ICC) analyses were used to identify time-associated differences and temporally stable CpGs, respectively. RESULTS: Around 60% of all EPIC array loci presented poor intra-individual stability (ICC <0.50); 78.114 (≈9%) showed good (ICC, 0.75-0.89), and 41.274 (≈5%) showed excellent (ICC ≥0.90) stability, between both measured time points. Focusing on previously identified consistently differentially methylated positions indicated that 22 CD-, 11 UC-, and 24 IBD-associated loci demonstrated high stability (ICC ≥0.75) over time; of these, we observed a marked stability of CpG loci associated to the HLA genes. CONCLUSIONS: Our data provide insight into the long-term stability of the PBL DNA methylome within an IBD context, facilitating the selection of biologically relevant and robust IBD-associated epigenetic biomarkers with increased potential for independent validation. These data also have potential implications in understanding disease pathogenesis.

Benefit of Risk-stratified Prophylactic Treatment on Clinical Outcome in Postoperative Crohn's Disease.

BACKGROUND AND AIMS: Whereas immediate postoperative treatment has shown effectiveness in reducing endoscopic postoperative recurrence [POR], evidence regarding the clinical benefit is limited. We compared rates of clinical POR in Crohn's disease [CD] patients receiving immediate prophylactic treatment with rates in patients receiving endoscopy-driven treatment. METHODS: We retrospectively collected data from 376 consecutive CD patients who underwent an ileocaecal resection with anastomosis between 2007 and 2018 with at least 3 years of follow-up at three sites. Subsequently, high- and low-risk patients categorised by established guidelines, who underwent endoscopy within 12 months postoperatively, were grouped according to a prophylactic- or endoscopy-driven approach and compared for incidence and time till endoscopic and clinical POR. RESULTS: Prophylactic treatment reduced rates of and time till endoscopic POR within 1 year in high-risk (hazard ratio [HR] 0.48, 95% confidence interval [CI] 0.27-0.86, p = 0.04, number needed to treat [NNT] = 5) but not low-risk [HR 0.90, 95% CI 0.32-2.56, p = 0.85] patients. Conversely, no significant differences in clinical POR within 3 years between prophylactic- and endoscopy-driven low-risk [HR 1.17, 95% CI 0.41-3.29, p = 0.75] and high-risk patients were observed [HR 1.06, 95% CI 0.63-1.79, p = 0.82, NNT = 22]. However, a large numerical albeit not statistical significant difference in 3-year clinical POR [28.6% vs. 62.5%, p = 0.11] in a subset of high-risk patients with three or more ECCO-defined risk factors was observed, indicating a cumulative effect of having multiple risk factors. CONCLUSION: Our observations favour step-up treatment guided by early endoscopic evaluation with prophylactic treatment reserved for carefully selected high-risk patients, in order to avoid potential overtreatment of a significant number of patients.

Novel DNA methylome biomarkers associated with adalimumab response in rheumatoid arthritis patients.

Background and aims: Rheumatoid arthritis (RA) patients are currently treated with biological agents mostly aimed at cytokine blockade, such as tumor necrosis factor-alpha (TNFα). Currently, there are no biomarkers to predict therapy response to these agents. Here, we aimed to predict response to adalimumab (ADA) treatment in RA patients using DNA methylation in peripheral blood (PBL). Methods: DNA methylation profiling on whole peripheral blood from 92 RA patients before the start of ADA treatment was determined using Illumina HumanMethylationEPIC BeadChip array. After 6 months, treatment response was assessed according to the European Alliance of Associations for Rheumatology (EULAR) criteria for disease activity. Patients were classified as responders (Disease Activity Score in 28 Joints (DAS28) < 3.2 or decrease of 1.2 points) or as non-responders (DAS28 > 5.1 or decrease of less than 0.6 points). Machine learning models were built through stability-selected gradient boosting to predict response prior to ADA treatment with predictor DNA methylation markers. Results: Of the 94 RA patients, we classified 49 and 43 patients as responders and non-responders, respectively. We were capable of differentiating responders from non-responders with a high performance (area under the curve (AUC) 0.76) using a panel of 27 CpGs. These classifier CpGs are annotated to genes involved in immunological and pathophysiological pathways related to RA such as T-cell signaling, B-cell pathology, and angiogenesis. Conclusion: Our findings indicate that the DNA methylome of PBL provides discriminative capabilities in discerning responders and non-responders to ADA treatment and may therefore serve as a tool for therapy prediction.

A BET Protein Inhibitor Targeting Mononuclear Myeloid Cells Affects Specific Inflammatory Mediators and Pathways in Crohn's Disease.

BACKGROUND: Myeloid cells are critical determinants of the sustained inflammation in Crohn's Disease (CD). Targeting such cells may be an effective therapeutic approach for refractory CD patients. Bromodomain and extra-terminal domain protein inhibitors (iBET) are potent anti-inflammatory agents; however, they also possess wide-ranging toxicities. In the current study, we make use of a BET inhibitor containing an esterase sensitive motif (ESM-iBET), which is cleaved by carboxylesterase-1 (CES1), a highly expressed esterase in mononuclear myeloid cells. METHODS: We profiled CES1 protein expression in the intestinal biopsies, peripheral blood, and CD fistula tract (fCD) cells of CD patients using mass cytometry. The anti-inflammatory effect of ESM-iBET or its control (iBET) were evaluated in healthy donor CD14+ monocytes and fCD cells, using cytometric beads assay or RNA-sequencing. RESULTS: CES1 was specifically expressed in monocyte, macrophage, and dendritic cell populations in the intestinal tissue, peripheral blood, and fCD cells of CD patients. ESM-iBET inhibited IL1ß, IL6, and TNFα secretion from healthy donor CD14+ monocytes and fCD immune cells, with 10- to 26-fold more potency over iBET in isolated CD14+ monocytes. Transcriptomic analysis revealed that ESM-iBET inhibited multiple inflammatory pathways, including TNF, JAK-STAT, NF-kB, NOD2, and AKT signaling, with superior potency over iBET. CONCLUSIONS: We demonstrate specific CES1 expression in mononuclear myeloid cell subsets in peripheral blood and inflamed tissues of CD patients. We report that low dose ESM-iBET accumulates in CES1-expressing cells and exerts robust anti-inflammatory effects, which could be beneficial in refractory CD patients.

Peripheral Blood DNA Methylation Profiles Do Not Predict Endoscopic Post-Operative Recurrence in Crohn's Disease Patients.

Prediction of endoscopic post-operative recurrence (POR) in Crohn's disease (CD) patients following ileocolonic resection (ICR) using clinical risk factors alone has thus far been inadequate. While peripheral blood leukocyte (PBL) DNA methylation has shown promise as a tool for predicting recurrence in cancer, no data in CD patients exists. Therefore, this study explored the association and predictive value of PBL DNA methylation in CD patients following ICR. From a cohort of 117 CD patients undergoing ICR, epigenome-wide PBL methylation profiles from 25 carefully selected patients presenting either clear endoscopic remission (n = 12) or severe recurrence (n = 13) were assessed using the Illumina MethylationEPIC (850K) array. No statistically significant differentially methylated positions (DMPs) or regions (DMRs) associated with endoscopic POR were identified (FDR p ≤ 0.05), further evidenced by the low accuracy (0.625) following elastic net classification analysis. Nonetheless, interrogating the most significant differences in methylation suggested POR-associated hypermethylation in the MBNL1, RAB29 and LEPR genes, respectively, which are involved in intestinal fibrosis, inflammation and wound healing. Notably, we observed a higher estimated proportion of monocytes in endoscopic POR compared to remission. Altogether, we observed limited differences in the genome-wide DNA methylome among CD patients with and without endoscopic POR. We therefore conclude that PBL DNA methylation is not a feasible predictive tool in post-operative CD.

Natural History and Risk Stratification of Recurrent Crohn's Disease After Ileocolonic Resection: A Multicenter Retrospective Cohort Study.

BACKGROUND: Prediction of endoscopic postoperative recurrence (POR) and prophylactic treatment based on clinical risk profile have thus far been inconclusive. This study aimed to examine the association between clinical risk profile and the development of endoscopic POR in a Crohn's disease population without postoperative treatment and to identify individual risk factors of endoscopic POR. METHODS: Medical records of 142 patients with Crohn's disease during follow-up after ileocecal or ileocolonic resection without prophylactic treatment at 3 referral centers were reviewed. Endoscopic POR was defined as a modified Rutgeerts score ≥i2b. Clinical risk profiles were distilled from current guidelines. Both uni- and multivariate logistic regression analysis were used to assess the relationship between risk profiles and endoscopic POR. RESULTS: Endoscopic POR was observed in 68 out of 142 (47.9%) patients. Active smoking postsurgery (odds ratio [OR], 3.01; 95% confidence interval [CI], 1.24-7.34; P = 0.02), a Montreal classification of A3 (OR, 3.05; 95% CI, 1.07-8.69; P = 0.04), and previous bowel resections (OR, 2.58; 95% CI, 1.07-6.22; P = 0.03) were significantly associated with endoscopic POR. No significant association was observed between endoscopic POR and any guideline defined as a high-/low-risk profile. However, patients with a combination of any 3 or more European Crohns & Colitis Organisation- (OR, 4.87; 95% CI, 1.30-18.29; P = 0.02) or British Society of Gastroenterology-defined (OR 3.16; 95% CI, 1.05-9.49; P = 0.04) risk factors showed increased odds of developing endoscopic POR. CONCLUSIONS: Our results suggest that patients with a combination of any 3 or more European Crohns & Colitis Organisation- or British Society of Gastroenterology-defined risk factors would probably benefit from immediate prophylactic treatment.

Deep Remission at 1 Year Prevents Progression of Early Crohn's Disease.

BACKGROUND & AIMS: We investigated the effects of inducing deep remission in patients with early Crohn's disease (CD). METHODS: We collected follow-up data from 122 patients (mean age, 31.2 ± 11.3 y) with early, moderate to severe CD (median duration, 0.2 years; interquartile range, 0.1-0.5) who participated in the Effect of Tight Control Management on CD (CALM) study, at 31 sites, representing 50% of the original CALM patient population. Fifty percent of patients (n = 61) were randomly assigned to a tight control strategy (increased therapy based on fecal level of calprotectin, serum level of C-reactive protein, and symptoms), and 50% were assigned to conventional management. We categorized patients as those who were vs were not in deep remission (CD endoscopic index of severity scores below 4, with no deep ulcerations or steroid treatment, for 8 or more weeks) at the end of the follow-up period (median, 3.02 years; range, 0.05-6.26 years). The primary outcome was a composite of major adverse outcomes that indicate CD progression during the follow-up period: new internal fistulas or abscesses, strictures, perianal fistulas or abscesses, or hospitalization or surgery for CD. Kaplan-Meier and penalized Cox regression with bootstrapping were used to compare composite rates between patients who achieved or did not achieve remission at the end of the follow-up period. RESULTS: Major adverse outcomes were reported for 34 patients (27.9%) during the follow-up period. Significantly fewer patients in deep remission at the end of the CALM study had major adverse outcomes during the follow-up period (P = .01). When we adjusted for potential confounders, deep remission (adjusted hazard ratio, 0.19; 95% confidence interval, 0.07-0.31) was significantly associated with a lower risk of major adverse outcome. CONCLUSIONS: In an analysis of follow-up data from the CALM study, we associated induction of deep remission in early, moderate to severe CD with decreased risk of disease progression over a median time of 3 years, regardless of tight control or conventional management strategy.